Sporadic colon cancer refers to >90% of colon cancer in the US and other developed countries. We have shown that this can be modeled by feeding C57Bl/6 mice a new western- style diet (NWD1) that combines a number of nutrient risk factors for colon cancer in the US and other western countries: 1) the NWD is formulated on the basis of nutrient density to reflect the higher fat and lower calcium and vitamin D3 levels consumed by large segments of the population; 2) tumors develop in control C57Bl/6 mice fed the diet for 2/3 of their life-span, similar to the 2/3 of the lifespan (ie 50-60 years) before most human sporadic colon cancer develops; 3) tumors develop at the same incidence and frequency as in the general population; 4) elevating calcium and vitamin D3 in the diet to levels associated with lower risk for colon cancer in the human prevents tumor development. We reported that in C57Bl/6 mice fed the NWD1 for 6 months, well before tumors form, there are subtle but significant alterations in gene expression in the flat, histologically normal mucosa of the large and small intestines that significantly overlap with alterations caused by inheritance of a mutant Apc allele, and that can be prevented by elevating calcium and vitamin D3 (NWD2). Thus, the perturbations track with probability of tumor formation. Finally, we have used a novel method of transcriptional imaging to demonstrate that Apc is haploinsufficient in the Apc1638N/+ mouse for regulating the dynamics of intestinal cell maturation, a perturbation of intestinal homeostasis that may also be caused by dietary induced higher risk. Our hypothesis is that development of sporadic colon cancer is a stochastic process the probability of which is determined by adaptation of the intestinal mucosa to patterns of nutrient intake. Aim 1, determines how the higher risk diet alters the number and positional distribution of Lgr5+ intestinal stem cells, allocation of these cells to different lineages, the role of Wnt and Notch signaling and their interaction in perturbations of mucosal homeostasis, and if these are prevented by elevation of calcium and vitamin D3. These experiments make use of novel methodology, including: a mouse genetic strain in which intestinal stem cells are marked and that also allows lineage specific tracing of their progeny; a method of cell isolation as a function of cell position along the crypt-luminal axis; and unique imaging of transcriptional activation of specific genes at the single cell level in situ. Aim 2 employs a model that more closely recapitulates human sporadic colon cancer in that conditional/inducible mutation of the Apc gene will be used to determine how the diets fed for 6 months prime the mucosa for development of preneoplastic and neoplastic lesions upon introduction of heterozygous or homozygous Apc mutation later in the animal's life-span following adaptation to nutritional factors. Aim 3 extends our published gene expression data and recently generated metabolomic data suggesting that both dietary and genetic risk for intestinal cancer cause an early shift towards glycolysis in cells of the normal appearing mucosa, specifically in the progenitor/proliferating cell compartment, long before such shifts that are known to characterize colon tumors. In response to comments made in the prior review, we will inhibit glycolysis with 2-deoxy-D glucose and determine how this modulates the dietary effects on stem cells, cell maturation pathways, and lineage specific allocation, and on inflammatory responses. Aim 4 has been added, again in response to specific comments in the review of the original application. In this aim we characterize the effects of the diets on inflammation and cytokine production of the colon and small intestine. Moreover, based on our recent reports dissecting the role of IL12 in mediating an important regulatory cross-talk between macrophages and colon tumor epithelial cells, and our data that this cytokine is elevated by the NWD1, but that this is prevented by elevating calcium and vitamin D in NWD2, we will use a novel neutralizing monoclonal Ab to IL12 to determine whether this eliminates or modulates the effects of diet on tumor associated alterations in the mucosa.